The overall aim of the proposed research is to gain an understanding of the molecular basis of neuronal differentiation. Nerve growth factor (NGF) induced differentiation of a clonal cell line, PC12, will be used as a model system for neuronal differentiation. The approaches involve the asessment of NGF mediated specific protein phosphorylations as a means of eliciting effects in the target cell which are involved in the expression of the neuronal phenotype. These approaches include (A) the identification of the kinase system responsible for NGF mediated protein phosphorylation. The protein substrate specificity and site of phosphorylation within a protein by NGF-activated kinase will be determined and the kinase identified with respect to known cellular protein kinases. This will be done by the use of specific activators and inhibitors of known kinases in vivo and by the direct measurement of specific kinase activities in cell homogenates. (B) Characteristics of the mechanism of NGF receptor transduction of the response will be determined with respect to functional characterization of the two-receptor types, subcellular localization, the means of adenylate cyclase activation and phosphatidylinositol turnover. (C) The analysis of tyrosin hydroxylase phosphorylation and activation with respect to the relationship of phosphorylation site and kinetic parameters of enzyme activation, subcellular localization of the different phpsphorylated forms, and the role of this modification in synaptogenesis in vivo in rat. (D) The general and specific requirements for NGF induced protein phosphorylation in neuronal differentiation will be investigated by specifically activating and blocking cellular kinases to cause or block respectively NGF effects on differentiation. These studies are intended to provide a basis for the eventual understanding of the role of protein kinase systems in the growth and development of neurons, the establishment of different developmental pathways of neurons, and the regulation of axonal guidance.